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Aims/Hypothesis: The insulin secretion/insulin resistance (disposition) index (ΔI/ΔG÷IR) commonly is used as a measure of beta cell function (Δ=change from baseline). This relationship is curvilinear and becomes linear when log transformed. ΔI is determined by two variables: insulin secretory rate (ISR) and metabolic clearance of insulin (MCR I ). We postulated that the characteristic curvilinear relationship would be lost if Δ plasma C-peptide (instead of Δ plasma insulin) was plotted against insulin sensitivity.

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Methods: 441 IGT individuals from ACT NOW received an OGTT and were randomized to pioglitazone or placebo for 2.4 years.

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Results: Pioglitazone reduced IGT conversion to diabetes by 72% (p<0.0001). ΔI/ΔG vs Matsuda Index (MI) of insulin sensitivity showed the characteristic curvilinear relationship. However, when ΔCP/ΔG or ΔISR/ΔG was plotted against MI, the curvilinear relationship was completely lost. This discordance was explained by two distinct physiologic effects that altered plasma insulin response in opposite directions: (i) increased ISR and (ii) augmented MCR I. The net result was a decline in plasma insulin response to hyperglycemia during OGTT. These findings demonstrate a physiologic control mechanism wherein the increase in ISR ensures adequate insulin delivery into portal circulation to suppress HGP while delivering reduced but sufficient amount of insulin to peripheral tissues to maintain the pioglitazone-mediated improvement in insulin sensitivity without excessive hyperinsulinemia.

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Conclusions: These results demonstrate the validity of disposition index when relating plasma insulin response to insulin sensitivity, but underscore the pitfall of this index when drawing conclusions about beta cell function, since insulin secretion declined despite an increase in plasma insulin response.

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Background Binding of insulin receptor substrate proteins 1 and 2 (IRS1/2) to the insulin receptor (IR) is essential for the regulation of insulin sensitivity and energy homeostasis. However, the mechanism of IRS1/2 recruitment to the IR remains elusive. Here, we identify adaptor protein APPL1 as a critical molecule that promotes IRS1/2-IR interaction.

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APPL1 Mediates Insulin Signaling via a Direct Binding to Insulin Receptor To elucidate the molecular mechanism underlying APPL1 action, we examined whether altering APPL1 expression affects insulin-stimulated phosphorylation of IRβ, IRS1, and Akt in C2C12 myocytes. Overexpression (Figure 3A) or suppression of APPL1 (Figure 3B) potentiated or impaired, respectively, insulin-stimulated tyrosine phosphorylation of IRS1 and Akt Thr 308 phosphorylation in C2C12 myocytes. On the other hand, altering the expression levels of APPL1 had little effect on the tyrosine phosphorylation of IRβ (Figures 3A and 3B), confirming that APPL1 promotes insulin signaling at a site downstream of IRβ. Given that APPL1 contains several protein-protein interaction domains (Figure S3A) and acts as a scaffold protein in adiponectin signaling (Xin et al., 2011), we examined whether APPL1 interacts with insulin-signaling molecules. By in vitro pull-down studies, we found that glutathione S-transferase (GST) fused to the C terminus of APPL1 (GST-APPL1 CT ), but not GST alone or GST fused to the BAR domain of APPL1 (GST-APPL1 BAR ), interacted with IRβ (Figure 3C). To determine whether APPL1 binds directly to IRβ, we examined the interaction between affinity- purified IRβ (Figures S3B and S3C) and GST-APPL1 fusion proteins (Figure S3D). The affinity-purified IRβ interacted with the GST-APPL1 and the GST-APPL1 CT fusion proteins, but not with the GST alone or GST-APPL1 BAR fusion proteins (Figure 3D), indicating that APPL1 physically interacts with IRβ directly. In addition, endogenous IRβ was coimmunoprecipitated with endogenous APPL1 in C2C12 myotubes, and the interaction was stimulated by insulin (Figure 3E). Of note, insulin treatment had no effect on the interaction between IRβ and APPL1 in the in vitro pull-down assays (Figure 3C), suggesting that the stimulatory effect of insulin may be a result of insulin-stimulated posttranslational modification of APPL1 rather than IR in intact cells.Figure 3 Figures 3Figure S3Xin et al., 2011Figure 3Figures S3Figure S3Figure 3

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APPL1 Interacts with IRS1/2, and the Interaction Is Inhibited by Insulin To further characterize the interaction between APPL1 and IRS proteins, we performed in vitro binding studies. We found that the GST-APPL1, but not GST, interacted with IRS1 (Figure 5A). A truncation mapping study indicated that multiple domains of APPL1 (the BAR, PH, and PTB) are involved in the interaction of APPL1 with IRS1 (data not shown). Coimmunoprecipitation studies revealed that endogenous IRS1 interacted with endogenous APPL1 in C2C12 myotubes under the basal condition (Figure 5B). Interestingly, the interaction between APPL1 and IRS1 is inhibited by insulin stimulation (Figure 5B), which is opposite to the interaction between APPL1 and IRβ that is stimulated by insulin (Figure 3E).Figure 5 Figure 3

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The Binding of APPL1 to IRβ Is Necessary for the Stimulatory Effect of APPL1 on Insulin-Stimulated IRβ-IRS1/2 Interactions and Signaling We investigated whether APPL1 promotes insulin signaling by facilitating the recruitment of IRS1 onto IRβ, which is a critical step for the transduction of insulin signaling to downstream targets (Taniguchi et al., 2006). We found that overexpression of APPL1 greatly enhanced the insulin-stimulated IRS1 interaction with IRβ in C2C12 cells (Figure 5C). On the other hand, suppression of APPL1 expression markedly reduced the effect of insulin on the interaction (Figure 5D). Together, these data suggest that APPL1 may exert its insulin-sensitization function by promoting IRS1’s interaction with IRβ. To determine whether the binding to IRβ is necessary for the stimulatory effect of APPL1, we examined the effect of APPL1 ΔCC, which is unable to interact with IRβ (Figure S3F), on insulin signaling. Overexpression of APPL1 ΔCC had no stimulatory effect on the interaction of insulin-stimulated IRS1 with IRβ and Akt phosphorylation (Figure 5C), indicating that the binding of APPL1 to IRβ is essential for the insulin-sensitizing effect of APPL1.Taniguchi et al., 2006Figure 5 Figure S3Figure 5 APPL1 Coordinates the Interaction of IRβ with IRS1 To determine if the APPL1/IR/IRS1 complex is dynamically regulated by insulin, we examined the kinetics of insulin-induced APPL1/IRS1 dissociation and IRS1/IRβ interaction. Insulin stimulation led to a time-dependent dissociation between APPL1 and IRS1 concurrently with a time-dependent increase in IRS1 tyrosine phosphorylation and the interaction between IRβ and IRS1 (Figure 5E). Together, these results suggest that APPL1 may function as a carrier to bring IRS1 to IRβ in response to insulin stimulation.Figure 5

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APPL1 Ser 401 Phosphorylation Correlates with Insulin Sensitivity APPL1 contains several potential phosphorylation sites including Ser 151, Ser 401, Ser 427, and Ser 430 (Gant-Branum et al., 2010) that may be subjected to insulin- or adiponectin-stimulated phosphorylation. Among these potential phosphorylation sites, Ser 401 is highly conserved in APPL1 among different species, and this residue is absent in the corresponding location of its isoform APPL2 (Figure S4A); the latter does not interact with the IR (data not shown). To determine the potential roles of APPL1 phosphorylation, we generated a phosphospecific antibody to Ser 401 of APPL1 (Figure S4B). By western blot analysis using this antibody, we found that APPL1 phosphorylation at Ser 401 is rapidly stimulated by insulin in C2C12 cells (Figure 6A) and in mouse skeletal muscle tissues (Figure 6B). The insulin-stimulated APPL1 Ser 401 phosphorylation was significantly reduced in insulin target tissues of mice fed a high-fat diet compared to mice fed with normal chow, which was associated with an impaired PI3K signaling pathway (Figures 6C, S4C, and S4D). Together, these results indicate a correlation between APPL1 phosphorylation at Ser 401 and insulin sensitivity in vivo.Gant-Branum et al., 2010Figure S4 Figure 6 Figures 6S4

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Ser 401 Phosphorylation Regulates APPL1 Binding to IRβ and Dissociation with IRS1 To investigate the role of Ser 401 phosphorylation in mediating the insulin-sensitizing role of APPL1, we overexpressed RNAi-resistant wild-type and phosphorylation mutants (S401A and S401D) of APPL1 in APPL1-suppressed C2C12 cells. Consistent with the notion that the binding of APPL1 to IRβ sensitizes insulin signaling by acting at a site downstream of the IR (Figures 2A, 2B, and S2A), overexpression of either APPL1 S401A or APPL1 S401D had no effect on insulin-stimulated IRβ tyrosine phosphorylation (Figure 6D). However, replacing Ser 401 with alanine greatly impaired the ability of APPL1 to bind with IRβ in response to insulin stimulation, concurrent with a loss of response to insulin-stimulated dissociation from IRS1 and IRS2 (Figure 6D). Consequently, the promoting effect of APPL1 on insulin-stimulated tyrosine phosphorylation of IRS1/2, phosphorylation of Akt at both Thr 308 and Ser 473, and GSK3β activation was significantly reduced by mutating serine 401 of APPL1 to alanine (Figure 6D). Conversely, the S401D mutant of APPL1 showed high binding affinity to IRβ, which occurs concurrently with a reduced interaction with IRS proteins and enhanced insulin signaling compared to wild-type APPL1 (Figure 6D). Taken together, these results clearly demonstrate that phosphorylation at Ser 401 plays a key role in mediating the insulin-regulated binding of APPL1 with IRβ and dissociation with IRS proteins.Figures 2S2Figure 6 APPL1 has previously been shown to bind to inactive Akt (Mitsuuchi et al., 1999). Consistent with this finding, we found that APPL1 interacted with Akt under basal conditions (Figure 6D). Like IRS1/2, Akt dissociates from the APPL1-IRβ complex in response to insulin stimulation (Figure 6D). These results suggest that in addition to facilitating IRS1/2’s interaction with the IR, APPL1 also promotes Akt translocation to the plasma membrane for insulin-stimulated activation. Interestingly, neither S401A nor S401D mutant had any effect on APPL1/Akt interaction compared with wild-type protein (Figure 6D), suggesting that phosphorylation at Ser 401 is not essential for regulating APPL1/Akt binding.Mitsuuchi et al., 1999Figure 6

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Adiponectin Promotes Ser 401 Phosphorylation and APPL1-IRβ Interaction To determine whether adiponectin has an effect on APPL1 phosphorylation and interaction with IRβ, we treated C2C12 cells with adiponectin alone or together with insulin. Adiponectin treatment greatly stimulated APPL1 phosphorylation at Ser 401 and enhanced APPL1 interaction with IRβ (Figures 7A and S5A). No synergistic effect of adiponectin and insulin on APPL1 phosphorylation and its binding to IRβ was observed (Figure 7A), suggesting that a common mechanism may be used by insulin and adiponectin to induce APPL1 phosphorylation. In agreement with this view, treating C2C12 cells with the protein kinase C (PKC) inhibitor Gö6983, but not inhibitors for GSK3β (LiCl), mitogen-activated protein kinase kinase (PD98059), or PI3K (LY ), blocked both insulin- and adiponectin-induced Ser 401 phosphorylation (Figures S5B and S5C). Taken together with the findings that phorbol ester phorbol-12-myristate-13-acetate stimulated the phosphorylation (Figure S5D) and that the insulin- and adiponectin-stimulated phosphorylation was inhibited by Gö6976 (Figure S5E), a selective inhibitor of Ca 2+ -dependent PKC isoforms such as PKCα and PKCβI, it is conceivable that a conventional PKC isoform may mediate both insulin- and adiponectin-stimulated APPL1 phosphorylation at Ser 401.Figures 7S5Figure 7Figures S5Figure S5

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To investigate the role of adiponectin-stimulated APPL1 phosphorylation in regulating APPL1/IRβ interaction, we overexpressed RNAi-resistant wild-type and phosphorylation mutants (S401A and S401D) of APPL1 in APPL1- suppressed C2C12 cells. Replacing Ser 401 with alanine greatly impaired adiponectin-stimulated APPL1 binding with IRβ (Figure 7B). Conversely, the S401D mutant of APPL1 displayed an enhanced ability to bind with IRβ (Figure 7B). Taken together, these results indicate that phosphorylation at Ser 401 provides a key mechanism by which adiponectin regulates the binding of APPL1 with IRβ and IRS1/2.Figure 7 While insulin treatment triggered the dissociation between APPL1 and IRS1/2 (Figures 6D and 7A), we found that adiponectin had no effect on APPL1/IRS1 dissociation (Figure 7A). Time course studies also revealed that adiponectin treatment did not affect the interaction between IRS1 and APPL1 or IRβ (Figure S5F). These results are consistent with our previous finding that adiponectin by itself does not stimulate Akt phosphorylation in cells (Mao et al., 2006). In agreement with these results, adiponectin treatment alone did not stimulate the tyrosine phosphorylation of IRβ and IRS1 or the serine/threonine phosphorylation of Akt and GSK3β (Figure 7A).Figures 67Figure 7Figure S5Mao et al., 2006Figure 7

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Results APPL1 forms a complex with IRS1/2 under basal conditions, and this complex is then recruited to the IR in response to insulin or adiponectin stimulation. The interaction between APPL1 and IR depends on insulin- or adiponectin- stimulated APPL1 phosphorylation, which is greatly reduced in insulin target tissues in obese mice. appl1 deletion in mice consistently leads to systemic insulin resistance and a significant reduction in insulin-stimulated IRS1/2, but not IR, tyrosine phosphorylation, indicating that APPL1 sensitizes insulin signaling by acting at a site downstream of the IR.